The gastrointestinal tract houses more than a trillion bacteria and must discriminate innocuous antigens and commensal bacteria from pathogenic microbes. A detailed understanding of how the mucosal immune system directs ensuing tolerogenic or immunogenic responses is essential for generating effective mucosal vaccines and treatments for inflammatory bowel disease (IBD). Such discriminatory activities are performed in part by antigen-presenting cells (APCs), which dictate the differentiation and migratory pathways of responding lymphocytes. Previous research has focused on how secondary lymphoid organ (SLO) APCs induce T cell differentiation, which has led to the widely accepted model that steady-state intestinal CD4+ T cell differentiation occurs solely in the mesenteric lymph nodes (mLN). However, little is known regarding intestinal APC-mediated T cell differentiation. Over the past several years, in vitro evidence has emerged suggesting that intestinal APCs are endowed with the capacity to induce T regulatory (Treg) or Th17 responses, depending upon the type of APC and the context of stimulation. Our studies suggest that intestinal macrophages and dendritic cells (DCs) are distinct in their localization and ability to induce Treg and Th17 differentiation. Furthermore, recent findings from our lab indicate that CD4+ T cell differentiation in the steady- state may take place in situ in the intestine, and thus, the intestine may serve as a novel site for CD4+ T cell differentiation-holding important clinical implications as a previously undefined source of tolerogenic and pro- inflammatory CD4+ T cells. The overall goal of this proposal is to define the location of intestinal CD4+ T cell differentiation in vivo by evaluating mice void of SLO and/or specific intestinal APC populations. We will specifically determine whether SLO and intestinal macrophages or DCs are required for intestinal CD4+ T cell differentiation during homeostasis and intestinal inflammation. In addition to gaining insights into the complex nature of intestinal CD4+ T cell differentiation induced by intestinal APCs, it is hoped that these studies will provide new ideas for the development of agents that alter APC and/or T cell functions for use as immunomodulatory agents in the treatment of IBD. The proposed research project will serve as a framework for the applicant's training plan designed to integrate basic science research in mucosal immunobiology with the applicant's career goal of becoming a gastroenterology physician-scientist whose research focus will be on the development of novel biologic therapies for IBD. PUBLIC HEALTH RELEVANCE: The mucosal immunology field is heavily focused on investigating intestinal DCs and devotes far less attention and resources to understanding the collective antigen-presenting cell network-in particular intestinal macrophages. This proposal will investigate the requirements for secondary lymphoid organs and intestinal macrophages and DCs in intestinal CD4+ T cell differentiation by providing the boldest examination yet of the functional effects of these cells in vivo during homeostasis and intestinal inflammation. This work will aid in understanding the immunological dysregulation that leads to human inflammatory bowel disease and what cells and factors may be appropriate targets for immunotherapy.